In most undifferentiated mammalian cells, microtubule arrays are arranged radially and are composed of highly dynamic microtubules. In many migrating cells and differentiated cells, microtubule arrays become polarized by the formation of a subset of unusually stable microtubules (1-3). In migrating wound-edge cells, stabilized microtubules are selectively formed near the leading edge (2, 4-6). These stable microtubules have a long half-life (>1 h) and are capped at their plus ends (7, 8). Stabilized microtubules accumulate post-translationally modified tubulin, such as detyrosinated tubulin, in which the C-terminal tyrosine of a-tubulin is removed by tubulin carboxypeptidase (9). Stable detyrosinated microtubules (termed Glu-MTs after their C-terminal glutamate) can be distinguished from predominantly tyrosinated dynamic microtubules (Tyr-MTs) with antibodies (10). Stable Glu-MTs may function as specialized tracks for vesicle and cytoskeletal trafficking. Kinesin interacts preferentially with Glu-tubulin in vitro (11), and two kinesin-dependent processes—the recycling of endocytosed transferring (12) and the extension of vimentin intermediate filaments (13, 14)—depend on stable Glu-MTs.
Using wounded monolayers of serum-starved NIH-3T3 fibroblasts, a signaling pathway that regulates stable microtubule formation has been identified. LPA in serum induces polarized stable microtubule formation in wound-edge fibroblasts through the small GTPase Rho and its effector, the formin mDia (4, 5). Stable microtubules induced by LPA-Rho-mDia are the result of microtubule capture and plus-end stabilization, or capping (4, 5). Although this pathway is sufficient to induce stable microtubules in serum-starved adherent fibroblasts, integrin signals, through FAK and lipid rafts, are responsible for restricting the formation of stable microtubules to the leading edge (6).
How mDia induces stable microtubules is not well understood. mDia partially colocalizes with stable microtubules and binds to taxol-stabilized microtubules (5), but whether other proteins are involved in this stabilizing activity has not been explored. As microtubule stabilization occurs primarily at the leading edge and results from the capping of microtubule plus ends, it is possible that microtubule plus-end-binding proteins (‘tip proteins’) (15) may participate by targeting microtubules to cortical sites and/or contributing to plus-end capping. In budding yeast, an analogous process of microtubule capture occurs at bud sites and is regulated by Rho GTPases and the formin Bni1, the yeast orthologue of mDia (16, 17). Genetic and other studies have identified additional proteins in this process, including the microtubule tip proteins Bim1 (also known as Yeb1) and Kar9 (18-21). Kar9 binds to Bim1 and functions by linking microtubules to actin filaments through Myo2 (22, 23). Microtubules are directed by Myo2 and actin cables towards the bud, where they undergo controlled shrinkage while maintaining their attachment to the bud (18-21). In mammalian cells, EB1 is the orthologue of yeast Bim1 (24), but there is no direct orthologue of Kar9. Because EB1 interacts with the tumour suppressor APC24, and Kar9 and APC have a region of limited sequence homology, it has been proposed that APC may be a functional homologue of Kar9 (25).
EB1 and APC both bind to microtubules in vitro (26, 27) and when either is overexpressed in cells, they bundle and stabilize microtubules (26, 28-30). This is consistent with a role for these proteins in microtubule stabilization. However, the stabilization induced by the overexpressed proteins may result from microtubule bundling, which is not normally observed in cultured cells. It is not clear whether these proteins function in the endogenous regulatory pathway where microtubules are stabilized by a plus-end capture mechanism, as in yeast.